Project Summary/Abstract Resistance to chemotherapy and molecularly targeted therapies is a major problem facing current cancer research. The development of drug resistance could lead to chemotherapy failure and tumor relapse, resulting in reduced survival rates. In addition, despite the unprecedented success achieved in cancer immunotherapy over the past decades, the cancer-cell-intrinsic mechanisms of resistance result in low response rates of patients which remains a major challenge. Therefore, it is urgently necessary to develop novel therapy to address these problems. Recently, our laboratory, headed by my sponsor Dr. Bing Xu, developed anticancer nanomedicine based on enzyme-instructed self-assembly (EISA). The EISA is capable of selective inhibition of tumor without harming normal tissues due to its precise spatiotemporal control. Additionally, by targeting multiple targets, EISA hardly induces acquired drug resistance. Most importantly, combination therapy holds great promise for tumor prevention and treatment. The overall goal of this project is to overcome cancer drug resistance through the application of nanotechnology and combination with anticancer immunotherapy. To achieve this goal, we proposed two following specific aims: 1) During the F99 phase, I propose to develop anticancer nanomedicines based on EISA to target loss-of-function (downregulation) and endoplasmic reticulum (ER) in cancer cells for minimizing the drug resistance and side effect. We designed a two enzyme controlled assembly system to target the downregulation via the combination of enzyme-instructed assembly and disassembly and tested its anticancer efficacy and selectivity. We will also study the EISA in targeting ER for cancer inhibition. The ER targeting ability and dynamic distribution of the designed molecules will be tested. Moreover, we will elucidate the mechanisms of cancer cell death induced by ER targeting. 2) For the K00, the direction of my postdoctoral research will focus on improving the anti-tumor response of immunotherapy through the combination with EISA based nanomedicines. The proposed postdoctoral training will provide me with new insight and techniques in cancer immunology.